Biochemistry 1994, 33, 13199-13207
13199
Exploring the Microtubule-Binding Region of Bovine Microtubule-Associated Protein-2 (MAP-2): cDNA Sequencing, Bacterial Expression, and Site-Directed Mutagenesis+ Richard L. Coffey, John C. Joly,* Brian D. Cain, and Daniel L. Purich* Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Health Science Center, Gainesville, Florida 32610-0245 Received April 5, 1994; Revised Manuscript Received July 26, 1994@
ABSTRACT: A 1.1 kilobase fragment of bovine microtubule-associated protein-2 (MAP-2) cDNA coding
for bovine MAP-2 microtubule-binding region (MTBR)was sequenced. Relative to mouse, rat, and human MAP-2, we observed striking preservation of primary structure, even beyond the sequence and spacing of the three nonidentical peptide repeats responsible for microtubule-binding interactions. For further analysis of microtubule-MAP interactions using site-directed mutagenesis, we developed a bacterial expression system coding for the MT-binding fragment of MAP-2 starting at the thrombin cleavage site (position 1629) and continuing to the C-terminus. This MT-binding fragment was purified to homogeneity by taking advantage of the unusual heat-stability and isoelectric properties of this cytomatrix component. We found that the MT-binding domain readily promoted tubulin polymerization, and the critical tubulin concentration was reduced in the presence of this recombinant protein. Because a second repeated sequence analogue can promote tubulin polymerization as well as displace the MT-binding region of MAP-2, this study was designed to learn more about the importance of each repeated sequence in M T binding. Accordingly, we mutated the first and third sequences to resemble the second repeated sequence, thereby generating the mutants designated m12-m2-m3, m1-11132, and m12-m2-m32. These recombinant proteins bound with an affinity comparable to or slightly better than equal concentrations of wild-type MT-binding fragment. Likewise, when the first or third sequence was replaced by an exact copy of the second octadecapeptide repeat, there was little, if any, increase in binding affinity, as reflected in the ability of mutant MTbinding fragments to promote tubulin polymerization. We can thus conclude that the binding energies associated with each of the three second-sequence repeats were not additive, and these findings fortify the conclusion that the second sequence repeat plays a dominant role in microtubule binding.
Microtubule-associated protein-2, a cytomatrix component of neuronal dendritic processes, interacts with microtubules (MTs)’ and is thought to stabilize the neuronal cytoskeleton by forming cross-links between microtubules (Olmsted, 1986). A single MAP-2 gene appears to serve as a precursor for several different protein species that arise from alternative splicing of a common MAP-2 “A precursor. MAP-2ab are high-molecular-weight forms appearing in dendrites of adult neurons (Papandrikopoulou et al., 1989), and MAP2c is a low-molecular-weight counterpart containing the same extreme amino and carboxy termini found in the adult forms. MAP-2ab can be readily cleaved by serine proteases (Vallee & Borisy, 1977) into on N-terminal projection-arm domain and a C-terminal microtubule-binding region, the latter designated here as MAP-2 MTBR. Unlike chymotrypsin and trypsin, however, the arginine-specific protease thrombin produces stable limit digestion products after cleavage at the +This research was supported in part by U.S. Public Health Service Research Grant NIH GM-44823. * To whom correspondence should be addressed. Current address: Genentech, Inc., 460 Point San Bruno Blvd., South San Francisco, CA 94080. Abstract published in Advance ACS Abstracts, October 15, 1994. Abbreviations: MT, microtubule: MAP-2 MTBR, generic designation of the microtubule binding region; IPTG, isopropyl P-thiogalactopyranoside; MES, 2-(N-morpholino)ethanesulfonicacid; PIPES, piperazine-N,”-bis(2-ethanesulfonic acid); PCR, polymerase chain reaction.
*
@
protease-accessible hinge region of MAP-2 (Flynn et al., 1987). The MT-binding fragment is quite basic with an isoelectric point of about 10.3, and the projection-arm is acidic with a corresponding isoelectric point of approximately 4.8 (Flynn et al., 1987). The 203-amino acid residue MTbinding fragment displays the same apparent affinity as MAP-2ab for microtubules (Joly et al., 1989). This region includes a triad of regularly spaced nonidentical octadecapeptide repeats (Lewis et al., 1988), and analogues corresponding to the second-repeated sequence promote tubulin polymerization (Joly et al., 1989) and displace MAP-2 from assembled microtubules (Joly & Purich, 1990). Most of the biochemical characterization of MAP-2 has been achieved through characterization of the heat-stable bovine or porcine protein, while the amino acid sequence was deduced using mouse or rat MAP-2 cDNA (Lewis et al., 1988; Kindler et al., 1990). MAP-2 phosphorylation is known to alter MT assembly properties (Jameson & Caplow, 1980), and recent studies have shown that phosphorylation enhances the dynamic instability of brain microtubules in vitro (Raffaelli et al., 1992). Our interest in analyzing MAP interactions with microtubules through site-directed mutagenesis led us to isolate and sequence the corresponding 1.1 kilobase fragment of bovine brain MAP-2 cDNA. We also developed an efficient bacterial expression system and purification method to isolate this microtubule-binding region of MAP-2, which provide for further characterization of the
0006-2960/94/0433-13199$04.50/0 0 1994 American Chemical Society
13200 Biochemistry, Vol. 33, No. 45, 1994 microtubule-binding properties by using site-directed mutagenesis. This allowed us to obtain new insights regarding the interactions of the nonidentical peptide repeats with microtubules.
EXPERIMENTAL PROCEDURES Materials [a-32P]dATP, SDS, and sucrose were ICN products. Amersham, Inc., supplied the [a-35S]dATP and their “Oligonucleotide-Directedin vitro Mutagenesis System” (version 2.1). Reverse transcriptase was from Promega, and Klenow and T4 DNA ligases were from New England Biolabs. Restriction endonucleaseswere purchased from New England Biolabs or Promega. The bovine brain cDNA library was obtained from Clonetech, Inc., and the Sequenase (version 2.0)DNA sequencing kit was a U.S. Biochemicals product. Expression vector pETh-3b, a derivative of PET-3b described by Studier et ul. (1990), was a gift from Dr. Donald R. McCarty of the Department of Vegetable Crops, University of Florida. Chloramphenicol and acetate kinase were purchased from Boehringer-Mannheim. Ampicillin, isopropyl P-thiogalactopyranoside (IPTG), 2-(N-morpholino)ethanesulfonic acid (MES), piperazine-N,N-bis(2-ethanesulfonic acid) (PIPES), EGTA, MgCl2, dithiothreitol, protease inhibitors, lysozyme, Tris-HC1, trifluoroacetic acid, and GTP were from Sigma. Phosphocellulose-P11 was a Whatman product. Taxol was obtained from Dr. Matthew Suffness of the National Cancer Institute, Bethesda, MD. Methods Cloning and Sequencing the Bovine MAP-2 MT-Binding Region. The mouse cDNA sequence data of Lewis et al. (1988) served as the basis for preparing polymerase chain reaction (PCR) primers spanning the amino acid sequences 5359-5388 (sense-primer) and 5643-5665 (antisenseprimer). We employed standard molecular biological techniques (Sambrook et al., 1989). Bovine brain RNA was isolated and transcribed into cDNA with reverse transcriptase using random hexamer primers. The polymerase chain reaction was then camed out for 30 cycles, products were fractionated on agarose gels, and bands were observed at sizes corresponding to 300 base pairs. These were electroeluted and treated with mung-bean nuclease to remove singlestranded ends, then ligated into the SmaI site of M13mp19, and sequenced. A MAP-2 clone was identified and subsequently ratiolabeled for use as a probe in the screening of a 2Agtll bovine brain cDNA library. Approximately 300 000 plaques were initially screened using duplicate filter lifts. Positives were picked and plated; two additional rounds were then performed, and two sets of clones were plaque-purified. One clone contained a 1.1 kilobase insert after a A liquid lysate preparation and EcoRI digestion. This 1.1 kilobase fragment was introduced into pUC19 for subcloning and sequencing. The resulting plasmid, pJJ1, was grown in large quantity and digested with EcoRI, and the 1.1kilobase insert was purified from an agarose gel. Several subclones were generated by digestion with Hue111 and AZuI and subsequently sequenced. This approach allowed us to locate two unique internal restriction sites (BamHI and SphI) from which three fragments spanning the entire 1.1 kilobase MT-binding region were generated. These fragments were subcloned into
Coffey et al. both M13mp18 and M13mp19 and sequenced in both orientations. Vector Construction. Plasmid pJJl was digested with SacI and XbaI which served to remove a fragment between a SacI site in the 3’-untranslated region of the clone and an XbaI site in the pUC19 polylinker. A short synthetic doublestranded oligonucleotidewith SacI and XbaI ends was ligated into the vector to recreate a circular double-stranded plasmid lacking the EcoRI site at the 3’ end of the MAP-2 clone. The resulting plasmid was digested with EcoRI (at the 5’ end of the clone) and HincII (adjacent to the XbaI site in the pUC19 polyliner) to liberate the 1.1 kilobase MAP-2 clone. This was then ligated into the EcoRI and EcoRV sites in the polylinker of the expression vector pETh-3b (McCarty et al., 1991) which had been previously digested with SphI and PvuII, the ends made blunt with T4 DNA polymerase, and ligated. Removal of this SphI-PvuII fragment destroyed the SphI site and removed two PpuMI sites so that these sites in the MAP-2 cDNA fragment could be subsequently employed. The resulting plasmid, pRC3, allows for the expression of the entire 323-amino acid C-terminal fragment of MAP-2 fused to the first 11 amino acids of the T7-gene 10 product. Plasmid pRC7 expressing the C-terminal 203amino acid microtubule-binding domain, described by Joly et al. (1989), was prepared by digestion with PpuMI (at the thrombin cleavage site in the MAP-2 amino acid sequence) and NheI (adjacent to the start codon), rendering the ends blunt with Klenow, followed by ligation. The resulting sequence was in-frame and coded for the 203-amino acid C-terminal MAP-2 fragment preceded by the sequence MetAla-Arg (the arginine is found in the native MAP-2 sequence). Plasmids expressing the m12-m2-m3, ml-m2-m32, and m12m2-11132 mutants were created using the Amersham kit for oligonucleotide-directedmutagenesis. A BamHI-SphI fragment from the MAP-2 cDNA (containing the m2, m3, and part of the ml repeated sequences) was ligated into the polylinker of M13mp19 so that a single-stranded DNA template could be produced. Synthetic oligonucleotides complementary to the ml and m3 regions but containing the desired substitutions were used to carry out the mutagenesis. Before expression and purification of the mutant proteins, the resultant vectors were screened and sequenced to confirm that the desired mutants were made. Bacterial Expression and Protein Pur8cation. Expression plasmids were transformed into Escherichia coli BL21(DE3) and grown on LB plates containing 50 pg/mL ampicillin and 50 pg/mL chloramphenicol. A single colony was used to inoculate a 5 mL LB culture containing 50 pg/mL ampicillin and 50 pg/mL chloramphenicol and grown overnight at 37 “C. This culture was diluted into 500 mL of LB containing 50 pg/mL ampicillin and 50 pg/mL chloramphenicol and grown at 37 “C to an optical density of 0.4 (600 nm). IPTG was added at a concentration of 1 mM and growth continued for 2 h. Cells were harvested by centrifugation at 5000g for 5 min and resuspended in 20 mL of buffer (80 mM PIPES, 1 mM EGTA, and 1 mM MgC12, pH 6.8). They were pelleted again at 5000g for 5 min and resuspended in 4 mL of a buffer containing 100 mM Tris-HC1, pH 8.0, 50 mM NaC1, 1.0 mM MgC12, 1.0 mM dithiothreitol, 40 pg/ mL DNase, 40 pglmL RNase, 100 pg/mL N-tosyl-Lphenylalanine chloromethyl ketone, 100 pg/mL P-p-tosylL-lysine chloromethyl ketone, and 0.1 mM phenylmeth-
Biochemistry, Vol. 33, No. 45, 1994 13201
MAP-2 Microtubule-Binding Region anesulfonyl fluoride. This suspension was subjected to two passes through a French pressure cell at 950 atm. Lysozyme was added to 1 mg/mL, and the solution was incubated at room temperature for 15 min with occasional stirring. This fluid was then layered onto 12 mL of 25% sucrose (w/v) containing 100 mM Tris-HC1 (pH 8.0) and centrifuged at 14000g for 20 min at 20 "C. The resulting pellet containing insoluble inclusion bodies was rinsed with a 50 mM TrisHCl buffer (pH 8.0) and resuspended using a Dounce homogenizer in 1 mL of 50 mM Tris-HC1 (pH 8.0) buffer, containing 200 mM NaCl and 1 mM dithiothreitol. This suspension was then rapidly heated to a final temperature of 80 "C and held for 10 min to solubilize the heat-stable MAP-2 microtubule-binding fragment. Subsequent incubation on ice for 20 min and centrifugation at 400000g for 6 min served to remove most of the aggregated contaminating proteins. The supernatant predominantly contained intact MAP-2 MT-binding fragment, and this was passed through a 0.22 pm filter and subjected to reverse-phase chromatography using a Brownlee large-bore CIS reverse-phase column. A linear, two-step gradient from pure H20 to 36% (v/v) acetonitrile in water after 45 min, and to 50% (v/v) acetonitrile in water after 75 min, caused the pure product to elute at approximately 52 min after the gradient commenced. [Trifluoroacetic acid (0.1% v/v) was present throughout the gradient.] Fractions containing pure MAP-2 microtubule-binding domain were pooled and dried in a Savant Speedvac concentrator, with subsequent resuspension in 1 mL of MEM buffer (100 mM MES, 1 mM EGTA, and 1 mM MgC12, pH 6.8). The solution was aliquoted, frozen in liquid nitrogen, and stored at -70 "C for later use. Assembly Assays and Critical Concentration Measurements. Bovine brain microtubule protein and tubulin were prepared as outlined elsewhere (Joly & Purich, 1990). Tubulin polymerization experiments were carried out in PEM buffer (80 mM PIPES, 1 mM EGTA, and 1 mM MgC12, pH 6.8) with 1 mM GTP, and a GTP-regenerating system composed of 2 units/mL acetate kinase, and 10 mM acetyl phosphate. Tubulin (10 pM) was assembled with various concentrations of recombinant MT-binding fragment. The assembly process was monitored using a Beckman DU-2 spectrophotometer at 350 nm in a cuvette thermostated at 37 "C. Critical concentration determinations were made by assembling tubulin at 20 p M with 3.4 p M recombinant MTbinding fragment. After a plateau level of polymerization was reached, the apparent absorbance at 350 nm was recorded. Tubulin was diluted to 15, 10, and 5 p M in the presence of a constant level of MT-binding domain (3.4 pM). The absorbance was recorded at each dilution point only after steady-state polymerization was reached. Competitive Binding Assays. Tubulin (50 pM) was permitted to self-assemble at 37 "C for 20 min followed by the addition of 50 p M taxol for an additional 10 min at 37 "C (Joly er al., 1990). Microtubules were diluted to 2.5 p M in PEM buffer with 1.0 mM GTP, 10 pM taxol, 1.0 mM dithiothreitol, 10 p M recombinant MT-binding fragment, and the indicated amounts of competitor peptide. After 20 min incubation at 37 "C, samples were centrifuged in a Beckman Airfuge through a 20% (w/v) sucrose cushion at lOOOOOg for 30 min at 37 "C. Pellets and supernatants were analyzed by electrophoresis according to the procedure of Laemmli (1970) and visualized after Coomassie staining. MAP-2 MTBR in the pellet fractions was determined by densitometry.
Protein was determined by the Bradford (1976) method with bovine serum albumin used as a standard. SDS gel electrophoresis was performed by the Laemmli (1970) method.
RESULTS Bovine Brain MAP-2 cDNA Sequence and Deduced Amino Acid Sequence. To gain further insight concerning the subsite interactions of the three nonidentical repeated sequences and possibly other regions within the bovine brain MAP-2 microtubule-binding domain, we have utilized molecular cloning techniques to isolate a 1.1 kilobase region corresponding to the C-terminus of MAP-2. For this purpose, we screened a bovine brain cDNA library with a bovine probe obtained by PCR amplification of bovine brain cDNA using oligonucleotide primers based on the mouse brain MAP-2 cDNA sequence. This approach allowed us to avoid sequence artifacts arising from the PCR technique and to maximize hybridization by using the bovine probe with a bovine library. Because the cDNA library had an average insert size of 1-2 kilobases, and because the carboxy-terminal microtubule-bindingregion of MAP-2 only spans 600-700 base pairs, this strategy promised to provide us with an intact cDNA fragment corresponding to the entire MT-binding region. Indeed, as shown in Figure 1, dideoxy sequencing confirmed that this was the case, and our fragment contained the C-terminal-bindingdomain beginning at a position corresponding to amino acid 1509 in the murine sequence and extending past the stop codon into the 3'-untranslated region. We have previously demonstrated that a thrombin cleavage fragment of MAP-2 fully retains the tubule-binding properties of intact MAP-2, and we can now assign this sequence as that spanning the region from residues 1629 to 1828 of the mouse sequence. The exceptions are three additional amino acids (HTP) in the bovine sequence (1631- 1633), an alanine-for-glycine substitution at 1639, and a glycine-for-serine change at position 1788. The additional HTP sequence near the thrombin cleavage site was also confirmed by Edman microsequencing (Joly et al., 1989). Dingus et al. (1991) also carried out Edman analysis of a chymotrypsin cleavage fragment of bovine MAP-2, and their fragment can now be definitively localized at positions corresponding to amino acids 1526-1544. Most significantly, the three nonidentical repeated sequences which are boxed in Figure 1 are identical in bovine and murine sequences, attesting to the validity of earlier work using these sequences to investigate interactions of bovine brain MAP-2 with bovine brain microtubules (Joly et al., 1989; Joly & Purich, 1990). Bacterial Expression and Purification of the Bovine Brain MAP-2 MT-Binding Domain. To achieve efficient expression of the microtubule-binding domain of MAP-2 in E. coli, we utilized the pETh-3b expression vector. This plasmid has the advantage that protein expression is under the control of the T7 promoter, and expression is carried out in the E. coli vartiant BL21 (DE3) which contains an integrated copy of the T7 RNA polymerase gene under control of the ZacUV5 promoter (Studier et al., 1990). Accordingly, expression commences upon the addition of IPTG to the bacterial culture medium. As shown in Figure 2, the MT-binding fragment of MAP-2, migrating with a molecular weight of 28 000, can be readily purified to homogeneity in three steps. Lane 1 is the cell-free extract, and lane 2 illustrates the state of
13202 Biochemistry, Vol. 33, No. 45, 1994
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Coffey et al.
Pmjectiorr
MT-Binding
Ami
Regiort
Site
I
I
1509
1539
K D K V D G F-5 K A Q A Pl5-GF K Q A R K T T A AF'65-a - K- A Q A P 12,VL F K - - Q - - A - - K- -D-- K- -V-1 9 D- -0-{VR K T T A AlG A E SI GCTCAGGCTCCCAGTGTATTTWCAGGCA AAGGACAAAGTCTCTGATGGA ~ T ~ A C C A A G CGGAAAACG ACAGCAGCZRGTTTAXT~A S P E K R S S S--P--E--K--R--S
L L
P P
R R
AGCCCAGAGAAGCGCTCTTCTCTGCCGAGA
1569
1599
1629
-a
-
V
S
V
S
G G
D D
R I Z E N RlEfE N
S
F F
P S S I L P P R R G P S S I L P P R R G CCTTCCTCCATCCTTCCTCCTCGCCGAGGT
GTCTCAGGAGACAGAGATGAGAACTTC
R R
A A
T T
T T
R R
S S
E E
P P
I I
R R
R R
G G
K K
S S
G G
T T
S S
T T
S
P P
T T
S L N S S I S S A R S L N S S I S S A R TCTCTCAACAGTTCTATCTCTTCTGCGCGG
CGGACCACTCGGTCAGAGCCAATTCGCAGA
T P G S T A I T P G T P G S T A I T P G ACCCCTGGATCAACCGCCATCACTCCTGGC
G T P G T P S Y P R T P P S Y S S R T P G T P G T P S Y P R T P P S Y S S R T P ACCCCACCAAGCTATTCTTCACGCACCCCG GGCACTCC~GGAACCCCCAGCTACCCCAGG
GCAGGAAAAAGTGGCACTTCCACACCCACT
R T P P K S P A T P V P S E K K V A I I T P G T P K S l G I L .T--p G - - T - - p - - K - - S - J A C - I - - L -V-- - P - - S - - E - - K - - KV A I I R T P P K S P A T P CGCACGCCTCCGWTCTCCTGCTACTCCC ACCCCTG AACCCCCAAGTCG~CCATCTTG GTTCCCAGTGAGAAGAAAGTCGCCATTATA 0
C
UVI
0.15
40
pr) W
\
3 -. 3
0.10
W
a
0.00
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0.25
v
2
X
20
A
0
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0 0
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m ,2 m 2 m 3 m, m 2 m S 2
type
m2m2m3
m,m2m2
FIGURE9: Extent and rate of tubulin polymerization induced by mutant and wild-type forms of MAP-2 MTBR. (A) Wild-type and mutants with either the first or the third repeat changed to resemble the second repeat. (B) Wild-type and mutants with the first or third repeat replaced with an identical copy of the second repeat. Duplicate assembly runs were made for each at 37 "C with 10 pM tubulin and 1 pM MTBR. The error bars represent the range. Extent (indicated by the open bars) and rate (indicated by hatched bars) of tubulin polymerization are averages of these duplicates.
found that the wild-type binding fragment displayed weaker binding than MAP-2 MTBR[mlz-m~-m3]or MAP-2 MTBR[ml-mz-m32], and all three bound less strongly than the combined mutant MAP-2 MTBR[ml~-mz-m32].In terms of the concentration of peptide m i required to displace half of th binding fragment from taxol-stabilized microtubules, the wild-type and combined mutant MAP-2 MTBR[mlz-m2-m3~1 gave values of 0.13 and 0.6 mM, respectively. This degree of enhanced binding falls considerably short of that expected if all three repeated sequences bind simultaneously on a microtubule, and these data suggest that the second repeated sequence is the primary site of interaction. In support of this conclusion is our separate finding that complete substitution of the mz region with m3, using cassette mutagenesis, greatly reduced the extent of binding. Indeed, in assembly studies with comparable levels of wild-type and this mutant binding domain, we observed that the extents of assembly after 25 min in the presence of 10 p M tubulin were 0.205 and 0.028, respectively (R. L. Coffey and D. L. Punch, manuscript in preparation). The latter value is only slightly above that which we typically observe for tubulin selfassembly in the absence of microtubule-associated protein. Sloboda, Dentler, and Rosenbaum (1976) first demonstrated that microtubule-associated proteins can greatly stimulate tubule polymerization, presumably by stabilizing nucleation as well as by reducing the critical concentration for microtubule self-assembly. Both the rate of polymerization and also the extent of polymerization, respectively, can be used as indices for evaluating MAP interactions with microtubules. Thus, as an independent test of the contribution of each peptide repeat to MAP binding, we studied the ability of mutant forms of the MT-binding region to stimulate microtubule assembly. Shown in Figure 9A are the rates and extents of MAP-stimulated tubulin polymerization for wild type, MAP-2 MTBR[mlz-mz-m3], and MAP-2 MTBR[ml-m2-m32]. These data demonstrate that conversion of the first repeat or third repeat to resemble the second repeat has little effect on the ability of MAPS to stimulate assembly. Furthermore, we tested two additional mutant forms: the first, designated as MAP-2 MTBR[m~-mz-m3],has the first repeat replaced by an exact m2 octadecapeptide repeat; and the second mutant form, designated MAP-2 MTBR[ml-m2-
m3], has the third repeat replaced by an identical mz sequence. The findings presented in Figure 9B indicate that the position of additional second peptide repeats has some statistically distinguishable effect on the rate of tubulin polymerization, and future work will be needed to address whether this is an effect on nucleation or elongation kinetics. As stated earlier, if multiple binding of mz-like sequences occurred, one should anticipate that the additivity of free energies of binding should be reflected in a substantial multiplicative change in the apparent affinity of a MAP for microtubulebinding sites. Here, MAP mutants containing extra copies of the second repeat sequence would be expected to be far more effective in promotiong tubule assembly, and the data in Figure 9B clearly indicate that the contrary is true.
DISCUSSION In the present report, we describe the purification and preliminary characterization of bacterially expressed MAP-2 MT-binding region starting from the thrombin cleavage site (numbered 1629 in the mouse sequence) to the C-terminus. Our focus on the microtuble-binding region of this brainspecific MAP relates to a general interest in defining the subsite interaction that stabilize fibrous MAP binding to microtubules. It is noteworthy that this region retains all of the binding properties of adult MAP-2ab (Joly et al., 1989), and bacterial expression and purification of wild-type and mutant binding regions now open the way for a more detailed biochemical and structural inquiry. Likewise, these developments should facilitate future experiments with living cells to understand structure/function relationships affecting the molecular design of this neuronal cytomatrix protein. Earlier sequencing of mouse and rat MAP-2 cDNA, respectively, revealed that both proteins have highly conserved regions in their extreme amino and carboxyl regions (Lewis et al., 1988; Kindler et al., 1990), and our studies with bovine MAP-2, a protein from an evolutionarily more distant organism, indicate the remarkable preservation of sequence throughout the microtubule-binding domain. Indeed, from position 1538 to the C-terminus at position 1828, there are only two substitutions (Le., an D E at 1564 and a G/S at 1785) and a three amino acid HTP insert in the bovine sequence immediately following the thrombin cleavage site
13206 Biochemistry, Vol. 33, No. 45, 1994 at position 1629. This HTP insert is also present in the human MAP-2 sequence (Albala et al., 1993). Such extensive preservation of sequence has been observed in only a few cases; most notable is the HSP70 multigene family of heat shock proteins (Hendrick & Hartl, 1993), including the endoplasmic reticulum protein BiP and the major constitutively expressed hsc7O proteins. While these are arguably the most conserved sequences known, differing only by 1 or 2 substitutions in their 650 residues, our observations with the microtubule-binding region also point to strong evolutionary pressure to maintain structure in this region of MAP2. One might expect that such conservation reflects the stability against mutation displayed by other proteins with which MAP-2 interacts, and the tubulins are widely recognized for their evolutionary stability. Studies with MAP-2 and related work with tau and MAP-4 indicate that the tubule-binding domain is primarily confined to the repeated sequence region may allow tau to bundle microtubules. Of course, proteins other than tubulin may interact with MAP2, and in this context, the functional significance of the sequence preservation will require further investigation. Flynn et al. (1987), for example, observed that the MTbinding region binds to neurofilaments, and scanning mutagenesis studies may provide a means for defining the locus of this complexation reaction. The present investigation has also allowed us to study the microtubule-binding properties of site-specific mutant forms of the MAP-2 MT-binding region. We have found that mutations in the first and third repeated peptide sequences can lead to somewhat higher affinity microtubule binding when such sequences are made to resemble the m2 subsite. It should be noted, however, that Goode and Feinstein (1994) recently demonstrated the importance of so-called inter-repeat regions that intervene between each of the nonidentical repeats in tau protein, and future work must address the importance of corresponding structural elements in MAP-2. Furthermore, as we gain additional insight regarding MAPmicrotubule interactions by making other single or multiple point mutations in the second repeat, we may be able to enhance binding even further. Earlier studies demonstrated that the inclusion of three additional amino acids (RVK) to the synthetic octadecapeptide m2 greatly improved the ability of the analogue to stimulate microtubule assembly and to displace high-molecular-weight MAP-2 from microtubules (Joly & Purich, 1990). When one considers that the ml and m3 sequences correspondingly have QVQ and NVK following their sequences, it is reasonable to anticipate that the introduction of additional positive charge by replacement with RVK should also enhance binding to microtubules. Indeed, many earlier studies have demonstrated that ionic charge is likely to be an important driving force in the complexation of MAP-2 with microtubules (Bryan, 1976; Yamauchi & Purich, 1987). Such high-affhity mutant forms could be especially valuable in manipulating microtubule interactions in neurons where MAP-2 is thought to crosslink microtubules through the projection-arm region. All available evidence points to a dominant role of the second repeated sequence in MAP-2 binding to microtubules, which in Figure 6 is illustrated in terms of @-subunitbinding based on the findings of Cross et al. (1990). First, peptide analogues of this subsite promote tubulin polymerization, whereas analogues to the other repeats do not (Joly et al., 1989). Second, only m2 peptide analogues displace MAP-2
Coffey et al. (Joly & Purich, 1990). Third, Dingus et al. (1991) demonstrated that antibody binding to the third repeat does not interfere with MAP-2 binding to microtubules, indicating that the repeated sequence makes no significant contribution to the binding energy. Fourth, that replacement of m2 by the corresponding m3 sequence results in almost complete loss of ability to stimulate tubule asembly also accords with this conclusion. While improvement in the microtubule binding affinity can be accomplished by conversion of the first and third sequences to resemble m2, the modest gain can be accounted for by assuming that only one sequence is actually engaged in microtubule binding and that we have effectively increased the molarity of mz-like sequences by a factor of 3. If the m2 and the additional mz-like sequences had interacted at three MT sites simultaneously, the binding would be the summation of the individual energy (AGoveran) binding energies (AG,). The Gibbs equation [AGoverall = -nRT In K ] predicts that any additivity in binding energy will have an exponential effect on the observed value of K . Hence, if each additional repeat sequence interaction augmented the binding energy by only 50%, the observed effect on K would be a factor of 30. This was not observed in our experiments where we obtained an improved binding corresponding to a factor of 3-4 at most, corresponding to a very small incremental change in AGoveran. In sum, we are drawn to the single m2 interaction model in Figure 6, and additional work will be required to discem whether the flanking inter-repeat sequences contribute further to binding energy. Although this second repeat region is quite important in MAP-2 interactions with MTs, we cannot exclude the participation of other subsites within the microtubule-binding region. Experiments with protein kinase C phosphorylation of MAP-2 indicate that residues immediately preceding and following the second repeat must contribute to the overall binding interaction (Ainsztein & Purich, 1994). Indeed, phosphorylations at S 1703 (located in the first inter-repeat). S1711 (positioned in the second repeat), and $1728 (located in the second inter-repeat) are all involved in modulating the binding of MAP-2 to microtubules. These findings are in harmony with the conclusion of Butner and Kirschner (1991) that the binding energy for tau protein interactions with MTs is delocalized and derives from a series of weak interactions on the part of small groupings of amino acids. It should be noted that tau proteins and MAP-2 share many common features within the MTBR region, with the notable exception that the first nonidentical repeat in tau can itself promote tubulin polymerization while this is not the case for the MAP-2 first repeat. Butner and Kirschner (1991) suggest that the higher relative affinity of the first tau repeat indicates that is may serve to anchor tau protein to the microtubule while the more weakly interacting remaining repeats release about once each millisecond (if addition is diffusion-limited). Although their model suggests that tau protein may assume a variety of bound positions on the microtubule lattice, there is no direct experimental method currently available for establishing or refuting such a proposal. The availability of the MAP-2 microtubule-binding domain should now permit detailed characterization of the subsite interactions within the repeated peptide sequences as they relate to microtubule binding. This should also permit us to address such related issues as (a) the location of phos-
MAP-2 Microtubule-Binding Region phorylation sites that may be important in controlling MAP-2 interactions with microtubules and/or other cytomatrix components, (b) the nature of phospholipid interactions, particularly phosphatidylinositol to MAP-2 (Yamauchi & Purich, 1987, 1993), and (c) the nature of MAP-2 binding interactions with neurofilaments, which are thought to occur through the binding domain region (Flynn et al., 1987). We should also be able to utilize site-directed mutagenesis to rearrange the order and spacing of the three repeated sequences in the microtubule-binding region of MAP-2.
ACKNOWLEDGMENT We thank Alexandra Ainsztein for her invaluable assistance during the cDNA subcloning and sequencing.
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